Endostatin is a natural occurring anti-
angiogenic peptide and has been shown to inhibit
tumor lymphangiogenesis by suppressing the expression of
tumor-stimulating
growth factors. We have previously shown that
fibronectin alternative extra domain A (EDA) facilitates lymphangiogenesis of
colorectal tumors. Since it is known that EDA interacts with
integrin α9 in the lymphatic endothelial cells (LECs), we hypothesized that
endostatin may target EDA-
integrin α9 pathway to inhibit
colorectal tumor-induced lymphangiogenesis. To test this hypothesis, we examined the effect of
endostatin on EDA secreted by SW480
colorectal cancer cells and treated human LECs with different doses of
endostatin in the presence of conditional medium from SW480 cells. We found that
endostatin significantly reduced EDA secretion by SW480 cells and the expression of
integrin α9 in LECs. Immunofluorescence studies showed that EDA and
integrin α9 colocalized on the cell membrane of LECs and these colocalizations were dramatically reduced by
endostatin. Co-immunoprecipitation studies demonstrated that EDA interacted with
integrin α9 in LECs, and showed that
endostatin treatment inhibited the formation of EDA-
integrin α9 complex in LECs. Furthermore, we found that the arrangement and polarity of LEC cytoskeletons were destroyed by
endostatin substantially, leading to a reduced formation of tube-like structures of LECs and a suppressed chemotaxis of LECs toward SW480 cells. Consistently, EDA and
integrin α9 expressions as well as lymphangiogenesis were significantly suppressed by
endostatin in
colorectal cancer xenografts. In conclusion, our results suggest that
endostatin reduces
colorectal tumor-induced lymphangiogenesis, at least in part, by inhibiting EDA-
integrin α9 pathway.